The invention relates generally to plant molecular biology. In particular, the invention relates to methods for the identification of compounds that regulate plant growth and development through the modulation of magnesium chelatase expression or activity.
Magnesium chelatase (Mg-chelatase) is an enzyme involved in the synthesis of chlorophyll in plants and photosynthetic microorganisms, and of bacteriochlorophyll in photosynthetic bacteria. Specifically, Mg-chelatase catalyzes the ATP-dependent insertion of a magnesium ion into protoporphyrin IX, to form Mg-protoporphyrin IX. Protoporphyrin IX is the last common intermediate in chlorophyll, bacteriochlorophyll and heme biosynthesis. In all photosynthetic organisms studied to date, Mg-chelatase activity requires three subunits, which are commonly referred to as CHL D, CHL H and CHL I.
The Mg-chelatase reaction is thought to occur in two steps, preactivation and catalysis. Grafe et al. (1999) Proc Natl Acad Sci 96:1941-1946. According to the current model, in the preactivation step, a CHL D dimer associates with two CHL I monomers in the presence of Mg2+ and ATP. In the chelation step, which involves the hydrolysis of ATP, the CHL H subunits interact with the CHL D subunits, resulting in the insertion of Mg2+ into protoporphyrinogen IX. It is unclear whether the CHL I subunit remains associated with CHL D during the second step. Id.
The genes or cDNAs encoding Mg-chelatase subunits from a variety of plants and microorganisms have been cloned and expressed. In vitro Mg-chelatase activity has been demonstrated by reconstitution of the three subunits of tobacco Mg-chelatase. Mg-chelatase mutants have been identified in barley and tobacco. For example, several recessive lethal mutations have been isolated in each of the genes encoding the three barley Mg-chelatase subunits, Xantha-f, Xantha-g and Xantha-h. Henningsen et al. (1993) R Dan Acad Sci Lett Biol Skr 42:1-349; and Hansson et al. (1999) Plant Biol 96:1744-1749. A mutation resulting in a single amino acid residue substitution in the CHL I subunit of tobacco Mg-chelatase results in heterozygous plants with reduced levels of chlorophyll and a yellow-green phenotype, and homozygous yellow seedling lethals. Kjemtrump et al. (1998) Plant J 14:91-100. Antisense RNA expression of the Mg-chelatase CHL I or CHL H subunit of tobacco results in a decreased growth rate chlorophyll deficiency in transgenic tobacco plants. Papenbrock et al. (2000) Plant J 22:155-164; and Papenbrock et al. (2000)Plant Physiol 122:1161-1170. In Arabidopsis, a transposon insertion mutation in the CHL I subunit (chlorata-42), results in a fusion of the carboxy terminus of the CHL I subunit to a short open reading frame in the transposon. Chlorata-42 plants have a pale green phenotype. However, to date, no study has shown that Mg-chelatase is essential in Arabidopsis.
It has been suggested that inhibitors of plant Mg-chelatases may have potential herbicidal activity (German patent application DE 197 17 656). However, there are no herbicides that are known to act by modifying the activity of this enzyme. Accordingly, there is a need for assays that can be used to detect modulators of Mg-chelatase activity. Inhibitors of Mg-chelatase activity or expression have use as herbicides.
The present inventors have discovered that Mg-chelatase is essential for the growth of Arabidopsis. Specifically, the inhibition of Mg-chelatase CHL H gene expression in Arabidopsis seedlings results in varying levels of chlorosis (yellowing), significantly reduced growth and developmental abnormalities. Thus, Arabidopsis Mg-chelatase can be used as a target for the identification of herbicides. Accordingly, the present invention provides methods for the identification of compounds that modulate Arabidopsis Mg-chelatase expression or activity, comprising: contacting a compound with Arabidopsis Mg-chelatase, or a subunit thereof, and detecting the presence and/or absence of binding between said compound and said Mg-chelatase, or detecting a change in Mg-chelatase expression or activity.
The methods of the invention are useful for the identification of herbicides and other compounds that can modulate plant growth and development. In addition, the methods of the invention are useful for the identification of compounds that stimulate the expression or function of Mg-chelatase expression or function. Such compounds can be used to promote or manipulate plant growth and development.